Mutant alpha-amylases

ABSTRACT

The invention relates to a mutant α-amylase obtained by making replacement or deletion of at least one of amino acid residues such as the 167th Gln, 169th Tyr and 178th Ala in the amino acid sequence set forth in SEQ ID NO:1 in an α-amylase having said amino acid sequence, or an α-amylase having a homology of at least 70% to said amino acid sequence, a gene encoding the mutant α-amylase, a vector, transformed cells, a process for producing a mutant α-amylase, comprising culturing the transformed cells, and a detergent composition comprising the mutant α-amylase. The mutant α-amylase of the invention has excellent properties of high resistance to chelating agents, high specific activity in an alkaline region and excellent stability to heat, and is hence useful for detergents for automatic dish washer, laundry detergents and the like.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to mutant liquefying alkaline α-amylaseswhich have excellent heat resistance, and are particularly useful asenzymes for detergents, and genes thereof

2. Description of the Background Art

When an α-amylase [EC.3.2.1.1] is used as an enzyme for detergents, ithas heretofore been said that a liquefying alkaline α-amylase, which candecompose starch at random and is stable to alkali and also to bothchelating component and oxidation bleaching component, is preferred.However, in liquefying amylases, a calcium ion is generally importantfor maintaining the structure of the enzymes, and the stability thereofis lowered in the presence of a chelating agent. Besides, most of suchenzymes have had the optimum pH in a neutral to weakly acidic range.

Under the foregoing circumstances, the present inventors found thatenzymes produced by alkaliphilic Bacillus sp. KSM-K38 (FERM BP-6946) andBacillus sp. KSM-K36 (FERM BP-6945) strains isolated from soil do notshow the lowering of activity at all in the presence of a chelatingagent at a high concentration by which deactivation is recognized in theconventional liquefying α-amylases, and have resistance to surfactantsand oxidizing agents and that they have higher activity on the alkalineside compared with the conventional liquefying α-amylases and are usefulas enzymes for detergents (Japanese Patent Application No. 362487/1998.

However, said enzymes exhibit inactivation at a temperature of 50° C. orhigher, and so the heat resistance thereof have been somewhatinsufficient in view of the fact that cleaning of clothing and tablewareis generally conducted at about 10 to 60° C.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an α-amylase whichis a liquefying alkaline α-amylase that has high activity on thealkaline side and is stable to both chelating component and oxidationbleaching component, and has excellent heat resistance.

The present inventors have acquired various mutant enzymes as toliquefying alkaline α-amylases and investigated them. As a result, ithas been found that when a mutation is introduced into a specified aminoacid residue in the amino acid sequence (SEQ ID NO:1) of amylase derivedfrom KSM-K38, the heat resistance of the enzyme is improved withoutlosing its properties such as resistance to chelating agents andresistance to oxidizing agents and high specific activity in an alkalineregion, and that the heat resistance can be further improved bycombining such mutations.

According to the present invention, there is thus provided a mutantα-amylase obtained by making replacement or deletion of at least oneresidue of amino acid residues respectively corresponding to the 11thTyr, 16th Glu, 49th Asn, 84th Glu, 144th Ser, 167th Gln, 169th Tyr,178th Ala, 188th Glu, 190th Asn, 205th His and 209th Gln in the aminoacid sequence set forth in SEQ ID NO:1 in an α-amylase having said aminoacid sequence, or an α-amylase having a homology of at least 70% to saidamino acid sequence.

According to the present invention, there is also provided a mutantα-amylase obtained by making replacement of a sequence corresponding to11 to 100 amino acid residues from the amino terminal in the amino acidsequence set forth in SEQ ID NO:1 in an α-amylase having said amino acidsequence, or an α-amylase having a homology of at least 70% to saidamino acid sequence by an amino acid sequence of another liquefyingα-amylase corresponding to said sequence of the amino acid residues.

According to the present invention, there are further provided genesrespectively encoding these mutant α-amylases, vectors having each ofthe genes, cells transformed by such a vector, and a production processof these mutant α-amylases, comprising culturing the transformed cells.

According to the present invention, there is still further provided adetergent composition comprising any one of these mutant α-amylases.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will become apparent from the following description and theappended claims, taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 illustrates a method for preparing a recombinant plasmid for theproduction of α-amylases derived from KSM-K38 and KSM-AP1378 strains.

FIG. 2 illustrates a method for introducing a mutation into an α-amylasegene derived from the KSM-38 strain.

FIG. 3 illustrates a method for replacing an N-terminal sequence of the(α-amylase gene derived from the KSM-38 strain by an N-terminal regionof an α-amylase gene derived from the KSM-AP1378 strain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The mutant α-amylases according to the present invention are obtained bymutating a gene encoding a liquefying alkaline α-amylase having theamino acid sequence set forth in SEQ ID NO:1 or an amino acid sequencehaving a homology of at least 70% to said amino acid sequence. However,an example where heat resistance is improved by deletion and/orreplacement of an amino acid has also been conducted on the conventionalliquefying α-amylases. For example, an enzyme obtained by deletingresidues from the 177th Arg to the 178th Gly in an enzyme derived fromB. amyloliquefaciens (J. Biol. Chem., 264, 18933, 1989) and an enzymeobtained by replacing the 133rd His in an enzyme derived from B.licheniformis by Tyr (J. Biol. Chem., 265, 15481, 1990) have beenreported. However, the liquefying alkaline α-amylases used in thepresent invention have a low degree of amino acid homology with theconventional liquefying alkaline α-amylases. In these α-amylases, a sitecorresponding to the residues from the 177th Arg to the 178th Gly hasbeen already deleted, and the amino acid corresponding to the 133rd Hishas been already Tyr. Therefore, the examples of the conventionalenzymes cannot be always applied. More specifically, the mutations ofthe amino acid sequence for improving the heat resistance in the presentinvention are entirely different from the examples up to the date.

Examples of the liquefying alkaline α-amylases include an enzyme(Japanese Patent Application No. 362487/1998) derived from a Bacillussp. KSM-K38 (FERM BP-6946) strain separated from soil by the presentinventors and having the amino acid sequence of SEQ ID NO:1 and anenzyme (SEQ ID NO:4) (Japanese Patent Application No. 362487/1998)derived from Bacillus sp. KSM-K36 (FERM BP-6945) and having a homologyof about 95% to the amino acid sequence of SEQ ID NO:1. Incidentally,the homology of the amino acid sequence is calculated in accordance withthe Lipman-Pearson method (Science, 227, 1435, 1985).

In order to obtain the mutant α-amylase according to the presentinvention, a gene encoding a liquefying α-amylase is first cloned frommicroorganisms which produce said liquefying α-amylase. As a methodtherefor, a general gene recombination method may be used. For example,the method described in Japanese Patent Application Laid-Open No.336392/1996 may be used. Examples of the gene include those set forth inSEQ ID NO:3 and SEQ ID NO:5.

A mutation is then introduced into the gene thus obtained. As a methodtherefor, any method may be adopted so far as it is a method ofsite-specific mutation commonly performed. The mutation can beperformed, for example, by using a Site-Directed Mutagenesis SystemMutan-Super Express Km kit produced by Takara Shuzo Co., Ltd. Anoptional sequence of the gene may be replaced by a sequence of anothergene corresponding to the optional sequence by using the recombinant PCR(polymer chain reaction) method (PCR protocols, Academic Press, NewYork, 1990).

The mutation for improving the heat resistance in the present inventionis desirably a mutation in which an amino acid residue corresponding tothe 11th Tyr in the amino acid sequence set forth in SEQ ID NO:1 isreplaced by Phe, an amino acid residue corresponding to the 16th Glu byPro, an amino acid residue corresponding to the 49th Asn by Ser, anamino acid residue corresponding to the 84th Glu by Gln, an amino acidresidue corresponding to the 144th Ser by Pro, an amino acid residuecorresponding to the 167th Gln by Glu, an amino acid residuecorresponding to the 169th Tyr by Lys, an amino acid residuecorresponding to the 178th Ala by Gln, an amino acid residuecorresponding to the 188th Glu by Asp, an amino acid residuecorresponding to the 190th Asn by Phe, an amino acid residuecorresponding to the 205th His by Arg, or an amino acid residuecorresponding to the 209th Gln by Val.

The improvement of heat resistance can also be achieved by replacing anamino acid sequence corresponding to 11 to 100 amino acid residues fromthe amino terminal (Asp) in the amino acid sequence of SEQ ID NO:1according to the present invention, preferably a sequence correspondingto amino acid residues from the 1st Asp to the 19th Gly, by an aminoacid sequence of another liquefying α-amylase corresponding to saidsequence of the amino acid residues.

Examples of said another liquefying α-amylase used in the replacementinclude an enzyme having the amino acid sequence set forth in SEQ IDNO:2. A site of its amino acid sequence corresponding to said amino acidresidues from the 1st Asp to the 19th Gly is from the 1st His to the21st Gly. The enzyme is an liquefied α-amylase derived from a Bacillussp. KSM-AP1378 (FERM BP-3048) strain, and the sequence of the gene isdisclosed in Japanese Patent Application Laid-Open No. 336392/1996.

In the mutant α-amylases according to the present invention, a mutationwith at least two kinds of replacement or deletion selected from thereplacement or deletion of the above-described various kinds of aminoacid residues and the replacement of the amino acid sequences combinedwith each other is also effective, and mutant enzymes more improved inheat resistance can be obtained by such a combination. Morespecifically, examples of the combination of mutations include acombination of at least two of the replacement or deletion of thevarious kinds of amino acid residues, a combination of at least two ofthe replacement of the amino acid sequence, and a combination of atleast two of the replacement or deletion of the amino acid residues andthe replacement of the amino acid sequence. Preferably, at least twomutations may be suitably combined from among mutations in which anamino acid residue corresponding to the 49th Asn is replaced by Ser, anamino acid residue corresponding to the 167th Gln by Glu, an amino acidresidue corresponding to the 169th Tyr by Lys, an amino acid residuecorresponding to the 190th Asn by Phe, an amino acid residuecorresponding to the 205th His by Arg, and an amino acid residuecorresponding to the 209th Gln by Val, and a mutation in which an aminoacid sequence corresponding to amino acid residues from the 1st Asp tothe 19th Gly is replaced by an amino acid sequence from the 1st His tothe 21st Gly in the amino acid sequence set forth in SEQ ID NO:2.

Examples of the most preferred combination include a combination ofmutations in which an amino acid residue corresponding to the 49th Asnis replaced by Ser, an amino acid residue corresponding to the 167th Glnby Glu, an amino acid residue corresponding to the 169th Tyr by Lys, anamino acid residue corresponding to the 190th Asn by Phe, an amino acidresidue corresponding to the 205th His by Arg, and an amino acid residuecorresponding to the 209th Gln by Val, and a combination of a mutationin which an amino acid sequence corresponding to amino acid residuesfrom the 1st Asp to the 19th Gly is replaced by an amino acid sequencefrom the 1st His to the 21st Gly in the amino acid sequence set forth inSEQ ID NO:2 with a mutation in which an amino acid residue correspondingto the an amino acid residue corresponding to the 167th Gln by Glu, anamino acid residue corresponding to the 190th Asn by Phe, or an aminoacid residue corresponding to the 209th Gln by Val.

In addition, mutations for improving other properties than the heatresistance, for example, a mutation for more enhancing resistance tooxidizing agents, in which an amino acid residue corresponding to the107th Met is replaced by Leu, a mutation for enhancing the detergency ofa laundry detergent, in which an amino acid residue corresponding to the188th Glu is replaced by Ile, and/or the like may be combined with theabove-described mutations.

The thus-obtained mutant α-amylases according to the present inventionare improved in stability to heat without losing excellent properties ofhigh resistance to chelating agents, and high specific activity in analkaline region, and are hence useful for detergents for automatic dishwasher, laundry detergents and desizing agents for fibers.

Such detergents may comprise one or more enzymes selected fromdebranching enzymes (for example, pullulanase, isoamylase,neopullulanase, etc.), α-glycosidases, glucoamylases, proteases,cellulases, lipases, pectinases, protopectinases, pectic acid lyases,peroxidases, laccases and catalases in addition to the above-describedmutant α-amylases.

Further, surfactants such as anionic surfactants, amphotericsurfactants, nonionic surfactants and cationic surfactants, chelatingagents, alkalizing agents, inorganic salts, resoiling preventives,chlorine scavengers, reducing agents, bleaching agents, fluorescent dyesolubilizers, perfume bases, caking preventives, enzyme activators,antioxidants, preservatives, coloring matter, bluing agents, bleachingactivators, enzyme stabilizers, phase adjusters, etc., which arecommonly incorporated into the classical detergents, may beincorporated.

The detergent composition according to the present invention can beproduced by combining the above-described mutant α-amylases with thepublicly known detergent components described above in accordance with amethod known per se in the art. The form of the detergent compositionmay be suitably selected as necessary for the end application intended,and the detergent composition may be provided in the form of, forexample, liquid, powder or granules. The detergent composition accordingto the present invention can be used as a laundry detergent, bleachingdetergent, detergent for automatic dish washer, drain cleaner,artificial tooth cleaner or the like. In particular, it can preferablyused as a laundry detergent, bleaching detergent or detergent forautomatic dish washer.

The mutant α-amylases according to the present invention may be used ascompositions for liquefaction and saccharification of starch and be alsocaused to act on starch together with one or more enzymes selected fromglucoamylase, maltase, pullulanase, isoamylase, neopullulanase, etc.

The mutant α-amylases according to the present invention may also beused as desizing agent compositions for fibers by incorporating anenzyme such as pullulanase, isoamylase or neopullulanase.

EXAMPLES

Determination of Amylase Activity and Protein Content:

The amylase activity and protein content of each enzyme was determinedin accordance with the following respective methods.

The determination of amylase activity was conducted by the3,5-dinitrosalicylic acid method (DNS method). After a reaction wasconducted at 50° C. for 15 minutes in a reaction mixture with solublestarch contained in a 50 mM glycine buffer (pH: 10), reducing sugarformed was determined by the DNS method. With respect to the enzymaticactivity, the amount of the enzyme, which forms reducing sugarcorresponding to 1 μmol of glucose for 1 minute, was defined as 1 unit.

The protein content was determined by means of a Protein Assay Kitproduced by Bio-Rad Laboratories making use of bovine serum albumin as astandard.

Referential Example 1

Screening of Liquefying Alkaline Amylase:

Soil (about 0.5 g) was suspended in sterilized water and subjected to aheat treatment at 80° C. for 15 minutes. A supernatant of theheat-treated suspension was suitably diluted with sterilized water, andthe resultant dilute solution was coated on an agar medium (Medium A)for separation. Culture was then conducted at 30° C. for 2 days to formcolonies. Those on the peripheries of which transparent halo based onamylolysis had been formed were screened, and isolated asamylase-producing bacteria. Further, the thus-isolated bacteria wereinoculated on Medium B and subjected to aerobic shaking culture at 30°C. for 2 days. After the culture, the resistance performance to achelating agent (EDTA) of a supernatant centrifugally separated wasdetermined, and its optimum pH was further measured to screen theliquefying alkaline α-amylase-producing bacteria.

Bacillus sp. KSM-K38 (FERM BP-6946) and Bacillus sp. KSM-K36 (FERMBP-6945) strains were able to be obtained by the above-describedprocess. Medium A: Trypton 1.5% Soyton 0.5% Sodium chloride 0.5% Coloredstarch 0.5% Agar 1.5% Na₂CO₃ 0.5% (pH 10) Medium B: Trypton 1.5% Soyton0.5% Sodium chloride 0.5% Soluble starch 1.0% Na₂CO₃ 0.5% (pH 10)

The mycological natures of the KSM-K38 and KSM-K36 strains are shown inTable 1. TABLE 1 KSM-K36 strain KSM-K38 strain (a) Results ofmicroscopic Bacili having sizes of 1.0-1.2 μm × 2.4-5.4 μm for K36observation stain and 1.0-1.2 μm × 1.8-3.8 μm for K38 strain. Ovalendospores (1.0-1.2 μm × 1.2-1.4 μm) are formed at near end or thecenter thereof. Having periplasmic flagella and motility. Gram stainingis positive. Having no acid-fast. (b) Growth state on various media:Incidentally, the strains are alkaliphilic and so 0.5% sodium carbonatewas added to media used in the following tests. Nutrient agar plateculture Good growth state. Form Good growth state. Form of of coloniesis circular. colonies is circular. Smooth surface and rough Smoothsurface and smooth periphery. Color of periphery. Color of coloniescolonies is pale-ocher. is yellowish-brown. Nutrient agar slant cultureGrown. Grown. Nutrient broth liquid culture Grown. Grown. Nutrient brothgelatin stab Good growth state. No Good growth state. No culture gelatinliquefaction is gelatin liquefaction is observed. observed. Litmus milkmedium Not changed. Not changed. (c) Physiological nature: Reduction ofnitrate and Reduction of nitrate is Reduction of nitrate isdenitrification positive. Denitrification positive. Denitrification isnegative. is negative. MR test Failed to judge because Failed to judgebecause the medium is alkaline. the medium is alkaline. V-P testNegative. Negative. Formation of indole Negative. Negative. Formation ofhydrogen sulfide Negative. Negative. Hydrolysis of starch Negative.Negative. Citrate utilization Grown on Christensen Grown on Christensenmedium but not grown on medium but not grown on Cocer and Simmons media.Cocer and Simmons media. Utilization of inorganic Nitrate is utilized,but Nitrate is utilized, but nitrogen source ammonium salt is notammonium salt is not utilized. utilized. Formation of pigment Formationof pale-yellow Negative. pigment on King B medium. Urease NegativeNegative Oxidase Negative Negative Catalase Negative Negative Range ofgrowth Temperature range for Temperature range for growth is 15-40° C.,and growth is 15-40° C., and optimum temperature range optimumtemperature for for growth is 30-37° C. pH growth is 30° C. pH rangerange for growth is pH for growth is pH 9.0-11.0, 8.0-11.0, and optimumpH for and optimum pH for growth is growth is pH 10.0-11.0. the same asdescribed above. Behavior against oxygen Aerobic. Aerobic. O—F test Notgrown. Not grown. Sugar utilization D-galactose, D-xylose, L-arabinose,lactose, glycerol, melibiose, ribose, D-glucose, D-mannose, maltose,sucrose, trehalose, D-mannit, starch, raffinose and D-fructose areutilized. Growth on salt-containing Grown at a salt concentration of12%, but not grown at medium a concentration of 15%.

Reference Example 2

Culture of KSM-K38 and KSM-K36 Strains:

The KSM-K38 or KSM-K36 strain was inoculated on the liquid medium B usedin Referential Example 1 to conduct shaking culture at 30° C. for 2days. The amylase activity (at pH 8.5) of a supernatant centrifugallyseparated was determined. As a result, these strains had activities of557 U and 1177 U per liter of the medium, respectively.

Referential Example 3

Purification of Liquefying Alkaline Amylase:

Ammonium sulfate was added to the resultant culture supernatant of theKSM-38 strain obtained in Referential Example 2 to 80% saturation. Afterstirring the resultant mixture, precipitate formed was collected anddissolved in 10 mM Tris-hydrochloride buffer (pH: 7.5) containing 2 mMCaCl₂ and dialyzed overnight against the same buffer. The dialyzate thusobtained was passed through a DEAE-Toyopearl 650M column equilibratedwith the same buffer and caused to be adsorbed on the column, and theintended enzyme was eluted with the same buffer by 0-1 M gradient ofsodium chloride concentration. After the active fraction was dialyzedagainst the same buffer, an active fraction obtained by gel filtrationcolumn chromatography was dialyzed against the above-described buffer,thereby obtaining a purified enzyme which gave a single band on bothpolyacrylamide gel electrophoresis (gel concentration: 10%) and sodiumdodecyl sulfate (SDS) electrophoresis. Incidentally, a purified enzymewas also able to be obtained from the culture supernatant of the KSM-K36strain in accordance with the same process as described above.

Reference Example 4

Properties of Enzyme:

(1) Action:

Both enzymes decompose the α-1,4-glycoside bonds of starch, amylose,amylopectin and partially decomposed products thereof and produceglucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4),maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7) fromamylose. However, the enzymes do not act on pullulan.

(2) pH Stability (Britton-Robinson's Buffer):

Both enzymes exhibit a residual activity of at least 70% in a pH rangeof 6.5 to 11 under treatment conditions of 40° C. and 30 minutes.

(3) Action Temperature Range and Optimum Action Temperature:

Both enzymes act in a wide temperature range of 20 to 80° C. and have anoptimum action temperature of 50 to 60° C.

(4) Temperature Stability:

Enzyme was incubated in a 50 mM glycine-sodium hydroxide buffer (pH: 10)at various temperature for 30 minutes and then residual anzymaticactivity was measured. As a result, both-enzymes showed a residualactivity of at least 80% at 40° C. and a residual activity of about 60%even at 45° C.

(5) Molecular Weight:

Both enzymes have a molecular weight of 55,000 ±5,000 as measured bysodium dodecyl sulfate polyacrylamide gel electrophoresis.

(6) Isoelectric Point:

Both enzymes have an isoelectric point of about 4.2 as measured byisoelectric focusing.

(7) Influence of Surfactant:

Even when both enzymes are treated at pH 10 and 30° C. for 30 minutes ina 0.1% solution of each of various surfactants such as sodium linearalkylbenzenesulfonates, sodium alkylsulfates, sodium polyoxyethylenealkylsulfates, sodium α-olefinsulfonates, the sodium salts ofα-sulfonated fatty acid esters, sodium alkylsulfonates, SDS, soap andSoftanol, they scarcely undergo inhibition of their activities (residualactivity: at least 90%).

(8) Influence of Metal Salt:

Both enzymes were treated at pH 10 and 30° C. for 30 minutes with eachof various metal salts, thereby determining the influence thereof.

The K38 strain is inhibited by 1 mM Mn²⁺(inhibitory rate: about 75%) andsomewhat inhibited by both 1 mM Sr²⁺and Cd²⁺(inhibitory rate: 30 to40%).

Example 1 Cloning of Liquefying α-Amylase Gene

A chromosome DNA extracted from cells of the KSM-K38 strain inaccordance with the method by Saito & Miura (Biochim. Biophys. Acta, 72,619, 1961) was used as a template to amplify a gene fragment (about 1.5kb) encoding a liquefying alkaline α-amylase (hereinafter referred to as“K38AMY”) having an amino acid sequence set forth in SEQ ID NO:1 by PCRmaking use of primers K38US (SEQ ID NO: 19) and K38DH (SEQ ID NO: 20).The thus-amplified fragment was cleaved with a restriction enzyme Sal I,and then inserted into a Sal I-Sma I site of an expression vector pHSP64(Japanese Patent Application Laid-Open No. 217781/1994), therebypreparing a recombinant plasmid pHSP-K38 with a structural gene ofK38AMY bonded to a trailing end of a potent promoter derived from thealkaline cellulase gene of a Bacillus sp. KSM-64 (FERM P-10482) straincontained in pHSP64 (FIG. 1).

Similarly, a gene fragment (about 1.5 kb) encoding a liquefying alkalineα-amylase (hereinafter referred to as “LAMY”) having an amino acidsequence set forth in SEQ ID NO:2, which had been obtained by using achromosome DNA extracted from cells of a Bacillus sp. KSM-AP1378 (FERMBP-3048) strain (Japanese Patent Application Laid-Open No. 336392/1998)as a template, and amplified by PCR making use of primers LAUS (SEQ IDNO: 21) and LADH (SEQ ID NO: 22) was inserted into a Sal I-Sma I site ofan expression vector pHSP64 in the same manner as described above,thereby preparing a recombinant plasmid pHSP-LAMY (FIG. 1).

Example 2 Preparation of Mutant K38AMY Gene-1

A Site-Directed Mutagenesis System Mutan-Super Express Km Kit producedby Takara Shuzo Co., Ltd. was used for a site-specific mutation. Therecombinant plasmid pHSP-K38 obtained in Example 1 was first used as atemplate to conduct PCR making use of primers CLUBG (SEQ ID NO: 23) andK38DH (SEQ ID NO: 20), thereby amplifying a fragment of about 2.1 kbfrom the leading end of a potent promoter derived from the KSM-64 strainto the trailing end of the liquefying alkaline α-amylase gene. Thisfragment was inserted into a Sma I site of a plasmid vector pKF19kattached to the above kit to prepare a recombinant plasmid pKF19-K38 forintroduction of mutation (FIG. 2).

After various kinds of oligonucleotide primers for introduction ofsite-specific mutation respectively set forth in SEQ ID NO:6 to NO:15were 5′-phosphorylated with a T4 DNA kinase, each of the resultantproducts and pKF19-K38 were used to conduct a mutation-introducingreaction in accordance with a method described in the kit, and anEscherichia Coli MV1184 strain (Competent Cell MV1184, product of TakaraShuzo Co., Ltd.) was transformed with the resultant reaction product.Recombinant plasmids were extracted from the resultant transformants toconduct base sequence analysis, thereby confirming the mutation.

The mutation-introduced gene was made a template plasmid uponintroduction of a different mutation by inserting an expression promoterregion and a mutant K38AMY gene portion into the Sma I site of pKF19kagain, thereby introducing another mutation in accordance with the sameprocess as described above.

Each of the thus-obtained mutant recombinant plasmids was used as atemplate to conduct PCR making use of primers CLUBG (SEQ ID NO: 23) andK38DH (SEQ ID NO: 20), thereby amplifying each of mutant K38AMY genefragments. This fragment was cleaved with a Sal I and then inserted intoa Sal I-Sma I site of an expression vector pHSP64 (Japanese PatentApplication Laid-Open No. 217781/1994) to prepare a plasmid forproduction of mutant K38AMY (FIG. 1).

Example 3 Preparation of Mutant K38AMY Gene-2 (Chimera with LAMY Gene)

Recombinant PCR was used for a mutation in which the N-terminal regionof the K38AMY gene is replaced by its corresponding region of an LAMYgene (FIG. 3). The recombinant plasmid pHSP-K38 obtained in Example 1was first used as a template to conduct PCR making use of primers K38DH(SEQ ID NO: 20) and LA-K38 (SEQ ID NO: 17), thereby amplifying afragment encoding a sequence from the 20th Gln to C-terminal of theamino acid sequence of K38AMY set forth in SEQ ID NO: 1. On the otherhand, the recombinant plasmid pHSP-LAMY was used as a template toconduct PCR making use of primers CLUBG (SEQ ID NO: 23) and LA-K38R (SEQID NO: 18), thereby amplifying a gene fragment encoding a sequence fromthe leading end of the potent promoter to the 21st Gly of the amino acidsequence of LAMY set forth in SEQ ID NO: 2. Second PCR making use ofboth DMA fragments, and primers CLUBG (SEQ ID NO: 23) and K38DH (SEQ IDNO: 20) was conducted, thereby amplifying a gene fragment (about 2.1 kb)encoding a substituted mutant enzyme (hereinafter abbreviated as“LA-K38AMY”) in which both fragments having respective complementarysequences derived from the primers LA-K38 (SEQ ID NO: 17) and LA-K38R(SEQ ID NO: 18) were bonded to the terminal, and a region encoding asequence from the 1st His to the 21st Gly of LAMY and successively aregion encoding a sequence from the 20th Gln to the C-terminal of K38AMYwere bonded to the trailing end of the potent promoter. This genefragment was cleaved with Sal I and inserted into a Sal I-Sma I site ofan expression vector pHSP64 (Japanese Patent Application Laid-Open No.217781/1994), thereby preparing a plasmid for production of mutantK38AMY (FIG. 1).

Example 4 Production of Mutant Liquefying Alkaline α-Amylase

Each of the various plasmids for production of mutant K38AMY obtained inExamples 2 and 3 was introduced into a Bacillus subtilis ISW 1214 strain(leuA metB5 hsdM1) in accordance with the protoplast method (Mol. Gen.Genet., 168, 111, 1979) to culture the resultant recombinant Bacillussubtilis at 30° C. for 3 days in a liquid medium (containing 8% of cornsteep liquor; 1% of meat extract; 0.02% of potassium primary phosphate;5% of maltose; 5 mM of calcium chloride; and 15 μg/mL of tetracycline).The resultant culture supernatant was dialyzed against a Tris-HCl buffer(pH: 7.0), and the dialyzate was caused to be adsorbed on aDEAE-Toyopearl 650M column equilibrated with the same buffer, and elutedby gradient of NaCl concentration. This eluate was dialyzed against a 10mM glycine buffer (pH: 10.0), thereby obtaining a purified enzyme ofeach mutant K38AMY.

Example 5 Assay of Heat Resistance-1

Purified preparations of an enzyme (abbreviated as “Y11F”) with the 11thTyr in SEQ ID NO:1 replaced by Phe, an enzyme (abbreviated as “N49S”)with the 49th Asn replaced by Ser, an enzyme (abbreviated as “E84Q”)with the 84th Glu replaced by Gln, an enzyme (abbreviated as “S144P”)with the 144th Ser replaced by Pro, an enzyme (abbreviated as “Q167E”)with the 167th Gln replaced by Glu, an enzyme (abbreviated as “Y169K”)with the 169th Tyr replaced by Lys, an enzyme (abbreviated as “A178Q”)with the 178th Ala replaced by Gln, an enzyme (abbreviated as “E188D”)with the 188th Glu replaced by Asp, an enzyme (abbreviated as “N190F”)with the 190th Asn replaced by Phe, and an enzyme (abbreviated as“Q209V”) with the 209th Gln replaced by Val were obtained in accordancewith the processes described in Examples 1, 2 and 4, and their heatresistance was assayed by the following method. As a control, wild typeK38AMY was used.

Each enzyme was added to a 50 mM glycine buffer (pH: 10.0) preincubatedat 50° C. so as to give a concentration of about 1.2 U/mL, and after 30minutes, the buffer was sampled to determine the residual amylaseactivity of the enzyme in accordance with the method described above inEXAMPLES. The activity of the enzyme at the start is regarded as 100% todetermine a relative activity, thereby regarding it as the residualamylase activity. The results are shown in Table 2. In the wild typeK38AMY, the residual activity was decreased to 15%, while all the mutantenzymes exhibited a high residual activity compared with the wild type.TABLE 2 Residual activity Enzyme (%) after 30 minutes Wild type 15 Y11F40 N49S 30 E84Q 25 S144P 30 Q167E 46 Y169K 63 A178Q 20 E188D 30 N190F 70Q209V 40

Example 6 Assay of Heat Resistance-2

Mutant enzymes with Q167E, Y169K, N19OF and Q209V among the mutationsdescribed in Example 5 combined in the following manner were prepared inaccordance with the processes described in Examples 1, 2 and 4.

-   -   Q167E/Y169K (abbreviated as “QEYK”, prepared by using primer of        SEQ ID NO: 16)    -   N190F/Q209V (abbreviated as “NFQV”)    -   Q167E/Y169K/N190F/Q209V (abbreviated as “QEYK/NFQV”)

With respect to these enzymes, the heat resistance was assayed by amethod similar to Example 5. However, the temperature in the heattreatment was changed to 55° C., and Q167E, Y169K, N190F and Q209V wereused as controls. As a result, as shown in Table 3, all the mutants wereobserved being improved in heat resistance by the combination, andQEYK/NFQV obtained by combining 4 mutations exhibited a residualactivity of 85% after 30 minutes even at 55° C. TABLE 3 Residualactivity Enzyme (%) after 30 minutes Q167E 7 Y169K 14 QEYK 45 N190F 20Q209V 1 NFQV 40 QEYK/NFQV 85

Example 7 Assay of Heat Resistance-3

The following mutant enzymes with the mutation NFQV described in Example6 combined with S144P described in Example 5, and further combined witha mutation of replacement of 16th Gln by Pro (abbreviated as “E16P”)were prepared in accordance with the processes described in Examples 1,2 and 4.

-   -   S144P/NFQV (abbreviated as “SP/NFQV”)    -   E16P/S144P/NFQV (abbreviated as “EPSP/NFQV”)

With respect to these enzymes, the heat resistance was assayed by amethod (50° C.) similar to Example 5. As a result, as shown in Table 4,improvement in heat resistance was observed by combining E16P withSP/NFQV. TABLE 4 Residual activity Enzyme (%) after 30 minutes SP/NFQV40 EPSP/NFQV 50

Example 8 Assay of Heat Resistance-4

The following mutant enzymes with QEYK/NFQV among the mutationsdescribed in Example 6 suitably combined with a mutation (abbreviated as“M107L”) with the 107th Met in SEQ ID NO:1 replaced by Leu, a mutation(abbreviated as “H205R”) with the 205th His replaced by Arg, and N49Samong the mutations described in Example 5 were prepared in accordancewith the processes described in Examples 1, 2 and 4.

-   -   M107L/QEYK/NFQV (abbreviated as “ML/QEYK/NFQV”)    -   N49S/M107L/QEYK/NFQV (abbreviated as “NSML/QEYK/NFQV”)    -   N49S/M107L/H205R/QEYK/NFQV (abbreviated as “NSMLHR/QEYK/NFQV”)

With respect to these enzymes, the heat resistance was assayed by amethod similar to Example 5. However, the temperature in the heattreatment was changed to 60° C.

As a result, heat resistance was additionally improved by combiningML/QEYK/NFQV with N49S, further H205R, and NSMLHR/QEYK/NFQV exhibited aresidual activity of 75% after 30 minutes even at 60° C. (Table 5) TABLE5 Residual activity Enzyme (%) after 30 minutes ML/QEYK/NFQV 30NSML/QEYK/NFQV 50 NSMLHR/QEYK/NFQV 75

Example 9 Assay of Heat Resistance-5

A mutant enzyme LA-K38AMY with a sequence from the 1st Asp to the 19thGly of K38AMY replaced by a sequence from the 1st His to the 21st Gly ofLAMY was obtained in accordance with the processes described in Examples1, 3 and 4. The heat resistance of this enzyme was assayed by the methoddescribed in Example 5. As a result, as shown in Table 6, improvement inheat resistance by the replacement was observed. TABLE 6 Residualactivity Enzyme (%) after 30 minutes Wild type 15 LA-K38AMY 33

Example 10 Assay of Heat Resistance-6

Into the gene of the mutant enzyme QEYK/NFQV described in Example 6, wasintroduced a mutation with a sequence from the 1st Asp to the 19th Glyreplaced by a sequence from the 1st His to the 21st Gly of LAMY inaccordance with the same processes as in Examples 1 and 3. With respectto a mutant enzyme LA-K38AMY/QEYK/NFQV obtained by using this enzyme inaccordance with the process described in Example 4, the heat resistancewas assayed by the same method (heat treatment temperature: 60° C.) asin Example 8.

As a result, heat resistance was additionally improved by thecombination, and LA-K38AMY/QEYK/NFQV exhibited a residual activity of63% after 30 minutes even at 60° C. (Table 7) TABLE 7 Residual activityEnzyme (%) after 30 minutes LA-K38AMY 1 QEYK/NFQV 40 LA-K38AMY/QEYK/NFQV63

Example 11 Detergent Composition for Automatic Dish Washer

A detergent composition for automatic dish washer was produced inaccordance with a formulation shown in Table 8, and various mutantenzymes were separately incorporated into this detergent composition toconduct a washing test. As a result, the mutant enzymes exhibited anexcellent detergent effect compared with the wild type enzyme when theenzymes having the same activity value as each other are added. TABLE 8Composition of detergent (%) Pluronic L-61 2.2 Sodium carbonate 24.7Sodium hydrogencarbonate 24.7 Sodium percarbonate 10.0 Sodium silicateNo. 1 12.0 Trisodium citrate 20.0 Polypropylene glycol 2.2 SiliconeKST-04 (product of Toshiba silicone Co., Ltd.) 0.2 Socarane CP-A45(product of BASF AG) 4.0

The mutant α-amylases according to the present invention have excellentproperties of high resistance to chelating agents, high specificactivity in an alkaline region, excellent stability to heat, and arehence useful for detergents for automatic dish washer, laundrydetergents, compositions for liquefaction and saccharification ofstarch, and desizing agents for fibers.

1. A mutant α-amylase obtained by making a substitution or deletion ofat least one amino acid residue of specific positions in SEQ ID NO:1, orby making a substitution or deletion of at least one amino acid residuecorresponding to the above-mentioned amino acid residue in a sequencehaving at least 70% homology to SEQ ID NO:1, wherein said at least oneamino acid residue is selected from the group consisting of: the 11^(th)Tyr, 16^(th) Glu, 49^(th) Asn, 84^(th) Glu, 144^(th) Ser, 167^(th) Gln,169^(th) Tyr, 178^(th) Ala, 188^(th) Glu, 190^(th) Asn, 205^(th) His and209^(th) Gln, and said mutant α-amylase possesses increased heatresistance and maintains resistance to chelating agents when compared toSEQ ID NO:1, and said mutant (α-amylase comprises an amino acid sequencewhich is at least 95% homologous to SEQ ID NO:1.
 2. A mutant α-amylaseobtained by making a substitution of an amino acid sequencecorresponding to 11 to 100 amino acid residues from the amino terminalAsp residue of the amino acid sequence set forth in SEQ ID NO:1 or anamino acid sequence corresponding to 11 to 100 amino acid residues fromthe amino terminal Asp residue of SEQ ID NO:1 of a sequence having atleast 70% homology to SEQ ID NO:1, with an amino acid sequencecorresponding to 11 to 100 amino acid residues from the amino terminalAsp residue of SEQ ID NO:2, wherein said mutant α-amylase possessesincreased heat resistance and maintains resistance to chelating agentswhen compared to SEQ ID NO:1.
 3. A mutant α-amylase obtained by making asubstitution of an amino terminal sequence from 1^(st) Asp through19^(th) Gly of SEQ ID NO:1 or an amino terminal sequence correspondingto 1^(st) Asp through 19^(th) Gly of SEQ ID NO:1 of a sequence having atleast 70% homology to SEQ ID NO:1, with an amino acid sequence from1^(st) His to 21^(st) Gly of SEQ ID NO:2, wherein said mutant α-amylasepossess increased heat resistance and maintains resistance to chelatingagents when compared to SEQ ID NO:1.
 4. A mutant (α-amylase obtained byintroducing a first mutation and a second mutation into SEQ ID NO:1 oran amino acid sequence having at least 70% homology to SEQ ID NO:1,wherein said first mutation consists of a substitution or a deletion ofat least one amino acid residue selected from the group consisting ofthe 11^(th) Tyr, 16^(th) Glu, 49^(th) Asn, 84^(th) Glu, 144^(th) Ser,167^(th) Gln, 169^(th) Tyr, 178^(th) Ala, 188^(th) Glu, 190^(th) Asn,205^(th) His and 209^(th) Gln, and wherein said second mutation consistsof a substitution of an amino acid sequence corresponding to 11 to 100amino acid residues from the amino terminal Asp residue of the aminoacid sequence set forth in SEQ ID NO:1, and wherein said mutantα-amylase possesses increased heat resistance and maintains resistanceto chelating agents when compared to SEQ ID NO:1.
 5. A mutant (α-amylaseobtained by introducing a first mutation and a second mutation into SEQID NO:1 or by making a substitution or deletion of at least one aminoacid residue corresponding to the above-mentioned amino acid residue inan amino acid sequence having at least 70% homology to SEQ ID NO:1,wherein said first mutation consists of: the substitution of an aminoacid residue selected from the group consisting of: the 11^(th) Tyr ofSEQ ID NO:1 with Phe, the 16^(th) Glu of SEQ ID NO:1 with Pro, the49^(th) Asn of SEQ ID NO:1 with Ser, the 167 Gln of SEQ ID NO:1 withGlu, the 169^(th) Tyr of SEQ ID NO:1 with Lys, the 190^(th) Asn of SEQID NO:1 with Phe, the 205^(th) His of SEQ ID NO:1 with Arg, and the209^(th) Gln of SEQ ID NO:1 with Val, and wherein said second mutationconsists of: substituting an amino terminal sequence from 1^(st) Aspthrough 19^(th) Gly of SEQ ID NO:1 with an amino acid sequence from1^(st) His to 21^(st) Gly of SEQ ID NO:2.
 6. A nucleotide sequenceencoding the mutant α-amylase according to claim 1 or a vectorcontaining said gene.
 7. A cell transformed by the vector according toclaim
 6. 8. A process for producing a mutant α-amylase, comprisingculturing the transformed cells according to claim
 7. 9. A detergentcomposition comprising the mutant α-amylase according to claim
 1. 10. Amutant α-amylase obtained by making a substitution or deletion of atleast one amino acid residue of specific positions in SEQ ID NO:1, or bymaking a substitution or deletion of at least one amino acid residuecorresponding to the above-mentioned amino acid residue in a sequencehaving at least 70% homology to SEQ ID NO:1, wherein said at least oneamino acid residue is selected from the group consisting of: the 11^(th)Tyr, 16^(th) Glu, 49^(th) Asn, 84^(th) Glu, 144^(th) Ser, 167^(th) Gin,169^(th) Tyr, 178^(th) Ala, 188^(th) Glu, 190^(th) Asn, 205^(th) His and209^(th) Gln, and wherein said mutant (α-amylase possesses increasedheat resistance, which is improved by combining mutations when comparedto SEQ ID NO:1, and maintains resistance to chelating agents andoxidizing agents when compared to SEQ ID NO:1, and said mutant(α-amylase comprises an amino acid sequence which is at least 95%.homologous to SEQ ID NO:1.
 11. A mutant (α-amylase obtained by making asubstitution or deletion of at least one amino acid residue of specificpositions in SEQ ID NO:1, or by making a substitution or deletion of atleast one amino acid residue corresponding to the above-mentioned aminoacid residue in a sequence having at least 70% homology to SEQ ID NO:1,wherein said at least one amino acid residue is selected from the groupconsisting of: the 11^(th) Tyr, 16^(th) Glu, 49^(th) Asn, 84^(th) Glu,144^(th) Ser, 167^(th) Gin, 169^(th) Tyr, 178^(th) Ala, 188^(th) Glu,190^(th) Asn, 205^(th) His and 209^(th) Gln, and wherein said mutantα-amylase: (i) possesses increased heat resistance when compared to SEQID NO:1; (ii) maintains resistance to chelating agents when compared toSEQ ID NO:1; (iii) maintains high specific activity under alkaline pHregion when compared to SEQ ID NO:1; and (iv) comprises an amino acidsequence which is at least 95% homologous to SEQ ID NO:1.
 12. The mutantα-amylase of claim 13, wherein said mutant α-amylase acts in an optimumtemperature range of 50° C. to 60° C.
 13. The mutant α-amylase accordingto claim 10, wherein the 11^(th) Tyr of SEQ ID NO:1 is substituted withPhe, the 16^(th) Glu of SEQ ID NO:1 is substituted with Pro, the 49^(th)Asn of SEQ ID NO:1 is substituted with Ser, the 167 Gln of SEQ ID NO:1is substituted with Glu, the 169^(th) Tyr of SEQ ID NO:1 is substitutedwith Lys, the 190^(th) Asn of SEQ ID NO:1 is substituted with Phe, the205^(th) His of SEQ ID NO:1 is substituted with Arg, and the 209^(th)Gln of SEQ ID NO:1 is substituted with Val.
 14. The mutant α-amylaseaccording to claim 11, wherein the 11^(th) Tyr of SEQ ID NO:1 isreplaced with Phe.
 15. The mutant α-amylase according to claim 11,wherein the 16^(th) Glu of SEQ ID NO:1 is replaced with Pro.
 16. Themutant α-amylase according to claim 11, wherein the 49^(th) Asn of SEQID NO:1 is replaced with Ser.
 17. The mutant α-amylase according toclaim 11, wherein the 167 Gln of SEQ ID NO:1 is replaced with Glu. 18.The mutant α-amylase according to claim 11, wherein the 169^(th) Tyr ofSEQ ID NO:1 is replaced with Lys.
 19. The mutant α-amylase according toclaim 11, wherein the 190^(th) Asn of SEQ ID NO:1 is replaced with Phe.20. The mutant α-amylase according to claim 13, wherein the 205^(th) Hisof SEQ ID NO:1 is replaced with Arg.
 21. The mutant α-amylase accordingto claim 13, wherein the 209^(th) Gln of SEQ ID NO:1 is replaced withVal.
 22. A mutant α-amylase obtained by making a substitution ordeletion of at least one amino acid residue of specific positions in SEQID NO:1, wherein said at least one amino acid residue is selected fromthe group consisting of: the 11^(th) Tyr, 16^(th) Glu, 49^(th) Asn,84^(th) Glu, 144^(th) Ser, 167^(th) Gln, 169^(th) Tyr, 178^(th) Ala,188^(th) Glu, 190^(th) Asn, 205^(th) His and 209^(th) Gln, and saidmutant α-amylase possesses increased heat resistance and maintainsresistance to chelating agents when compared to SEQ ID NO:1, and saidmutant α-amylase comprises an amino acid sequence which is at least 95%homologous to SEQ ID NO:1.
 23. A mutant (α-amylase obtained by making asubstitution or deletion of at least one amino acid residue of specificpositions in SEQ ID NO:4, wherein said at least one amino acid residueis selected from the group consisting of: the 11^(th) Tyr, 16^(th) Glu,49^(th) Asn, 84^(th) Glu, 167^(th) Gln, 169^(th) Tyr, 178^(th) Ala,188^(th) Glu, 190^(th) Asn, 205^(th) His and 209^(th) Gln, and saidmutant α-amylase possesses increased heat resistance and maintainsresistance to chelating agents when compared to SEQ ID NO:4, and saidmutant α-amylase comprises an amino acid sequence which is at least 95%homologous to SEQ ID NO:4.